Method of treating insulin resistance, adult onset diabetes and metabolic syndrome x

ABSTRACT

A method of treating insulin resistance, adult onset diabetes, and metabolic syndrome X and its related complications, in mammalian subject is accomplished by intravenously administering to a mammalian subject, a therapeutically effective amount of a liposomal suspension of lipoprotein small unilamellar vesicles (SUVs) comprising predominantly phospholipids. The liposomal suspension is administered over a period of time, whereby in the levels of some or all of blood glucose, insulin, total cholesterol, LDL cholesterol, triglyceride, creatine kinase (CK), creatine kinase-MB (CK-MB), Hb-A1 c , lipoprotein (a), SGOT and SGPT fall back within the normal range or are significantly reduced.

FIELD OF THE INVENTION

The present invention relates to a method of treating insulinresistance, adult onset diabetes, and metabolic syndrome X and itsrelated complications in a mammal using a therapeutic liposomalsuspension, comprising predominantly phospholipids.

BACKGROUND OF THE INVENTION

For maintenance of homeostasis, it is critical to keep a constant supplyof glucose to the cells to avoid the occurrence of dysglycemia and itsrelated metabolic imbalances. If left ignored or untreated, metabolicdysglycemia would lead to numerous metabolic diseases, includingobesity, heart disease, hypertension, diabetes, chronic fatigue,accelerated aging, degenerative disease, as well as many mental andemotional problems. For this reason, it is important to identify and, atthe same time, treat this condition early before costly and disablingdegenerative conditions arise that can ruin the quality of life as wellas shorten it.

Dysglycemia and diabetes show up very slowly, silently and dangerously.As disclosed by the American Diabetes Association, more than a third ofAmerican adults suffering from diabetes—more than five millionpeople—are not even aware that they have the disease. Many will learn oftheir condition only after they develop a severe debilitating disorder,such as heart disease, stroke, impaired vision, kidney disease, nervedamage or impotence. American Diabetes Association: Facts and Figures,2000, at www.diabetes.org/info/facts/facts_natl.jsp.

Diabetes or diabetes mellitus is a disease that occurs when the bodycannot make use of the glucose in the blood for energy because eitherthe pancreas is not able to make enough insulin or the insulin that isavailable is not effective. The early signs of diabetes are glucoseintolerance and insulin resistance. There are two main types of diabetesmellitus: insulin-dependent (type 1) and noninsulin-dependent (type 2 oradult onset diabetes).

A third type of diabetes is the gestational diabetes that develops onlyin pregnant women with no previous history of diabetes. Nearly 135,000U.S. women develop gestational diabetes each year. Typically,gestational diabetes clears up on its own after women have deliveredtheir babies. But studies show that about 40% of women with gestationaldiabetes go on to develop type 2 diabetes within 15 years (NIHPublication No. 02-3873, May 2002).

In insulin-dependent diabetes (IDDM; type I diabetes), the pancreasmakes little or no insulin because the insulin-producing beta cells havebeen destroyed. This usually appears at any age but usually occursbetween infancy and the late 30's, most typically in childhood oradolescence. Treatment consists of daily insulin injections or use of aninsulin pump, a planned diet and regular exercise, and dailyself-monitoring of blood glucose. If the level of insulin is too low fora long period of time, the body begins to break down its stores of fatfor energy. This causes the body to release fatty acids which are thenconverted into ketone bodies or ketoacids that are toxic at high levels.The result is called ketoacidosis, a severe condition that may put aperson into a coma if not treated right away.

In noninsulin-dependent diabetes (NIDDM; type II diabetes or adult onsetdiabetes (AOD)), the pancreas cannot produce enough insulin or the bodytissues become resistant to insulin. Because insulin is not available oris improperly used, the blood sugar level rises above the safety level.The patient's blood sugar level often rises gradually, taking severalyears to reach unsafe levels and cause symptoms. Thus, in some people,where the diabetic condition has not yet developed, normal or excessivelevels of insulin compensate for such resistance. Over time however,insulin production often drops and resistance worsens. About 90-95% ofall diabetic people have AOD. It is more common in people over the ageof 40.

AOD is caused by a complicated interplay of genes, environment, insulinabnormalities (reduced insulin secretion in the beta cells and insulinresistance in muscle cells), increased glucose production in the liver,increased fat breakdown, and possibly defective hormonal secretions inthe intestine. The recent dramatic increase of this disease indicatesthat lifestyle factors, such as obesity and sedentary lifestyle, may bestrong contributory factors in releasing the genetic elements that causethe disease.

Insulin-stimulated glucose uptake is widely variable among individuals(Stern, M. P. and Mitchell, B. D. Genetics of Insulin Resistance. In G.M. Reaven and A. Laws (eds). Insulin Resistance, The Metabolic SyndromeX, p. 3-18, Humana Press Inc., Totowa, N.J., 1999). The degree ofinsulin resistance observed in normal individuals can equal that seen indiabetic individuals. Hence, it is now widely known that insulinresistance precedes the development of adult onset diabetes. It is alsoequally essential to acknowledge that mild or even severe insulinresistance may be found in individuals who will never develop diabetes.Genetic factors contribute to this normal variation in insulinresistance. The common forms of insulin resistance include skeletalmuscle insulin resistance, hepatic insulin resistance and adipose tissueinsulin resistance. The entire contents of G. M. Reaven and A. Laws,(eds), Insulin Resistance, The Metabolic Syndrome X, p. 3-18, HumanaPress Inc., Totowa, N.J., 1999, are incorporated herein by reference intheir entirety.

The cellular response to insulin is mediated through a specific insulinreceptor in the plasma membrane. When insulin activates the receptor,the β-subunit is autophosphorylated at the juxtamembrane domain, thekinase domain, and the C-terminal domain. Full receptorautophosphorylation subsequently activates the protein tyrosine kinasereceptor activity, which together are necessary for the cellularresponse to insulin (Kahn, C. R. et al., J. Clin. Invest. 82:8622-8626,1988).

Insulin resistance is an impaired response to normal levels of exogenousor endogenous insulin in cells, tissues, the liver or the entire body.It can be caused by several factors, namely: (1) obesity factors (suchas elevated levels of free fatty acids and the association of insulinresistance with cytokines, e.g., resistin and leptin (Mooradian A. D.,Growth Horm. IGF Res. 11:Suppl A:S79-83, 2001; Ravussin, E. and Smith,S. R., Ann. N.Y. Acad. Sci. 967:363-78, 2002)); (2) proteins likecalpains (Baier, L. J. et al., J. Clin. Invest. 106:819-21, 2000); (3)abnormal regulation of amylin and calcitonin gene-related peptide (CGRP)that affect both the circulatory and nervous system (Leighton, B. andCooper, G. J., Nature 335:632-5, 1988; Haynes, J. M. et al. Diabetologia40:256-61, 1997); (4) elevated levels of interleukin 6 (IL-6) andC-reactive protein (CRP) that act as inflammatory and damage markers(Hak, A. E. et al., J. Clin. Endocrinol. Metab. 86:4398-405, 2001;Pickup, J. C. et al., Diabetologia 40:1286-92, 1997); and (5) increasedlevel of growth hormone during puberty (Dunger, D. B. and Cheetham, T.D., Horm. Res. 46:2-6, 1996; Halldin, M. U. et al., Clin. Endocrinol.(Oxf) 48:785-94, 1998).

The main cause of death in people with AOD, regardless of sex or age, isheart disease. Other complications associated with diabetes includenerve damage (neuropathy) and vascular abnormalities in both small andlarge blood vessels. Heart attacks account for 60% and stroke for 25% ofdeaths in all diabetics. People with diabetes are at risk for heart-riskconditions that include hypertension, high triglyceride levels and lowerhigh density lipoprotein, blood clotting problems, neuropathy, andsilent ischemia. To avoid some of these complications, diabetic patientsare treated with statins to improve their cholesterol and lipid levels,e.g., pravastatin (Pravachol), simvastatin (Zocor), fluvastatin(Lescol), atorvastatin (Lipitor), and rosuvastatin (Crestor). Niacin canalso be administered to improve the cholesterol profile but it alsoincreases blood sugar level.

Drug therapy is one common approach to treatment of adult onsetdiabetes. Oral agents such as sulfonylureas (e.g., glyburide, glipizide,glimepiride), meglitinides, biguanides, thiazolinediones, andalpha-glucosidase inhibitors, singly or combined, with or withoutinsulin replacement therapy are used currently.

Some forms of insulin analogues may be useful for patients having adultonset diabetes. However, the possible adverse effects of insulin onweight gain and the heart are troublesome. In fact, lower mortalityrates were obtained with drug treatment therapy (metformin (8%),sulfonylurea (16%) and thiazolinediones (14%) than insulin treatment(28%)).

Metabolic Syndrome X (MS-X) is a condition that promotes atherosclerosisand increases the risk of cardiovascular events through the collectionof independent and related complications or disorders. This conditionitself has been variously referred to as “syndrome X,” “insulinresistance syndrome” (Li, C. et al., Diabetes Care 24: 2035-2042, 2001),“Reaven's syndrome” (Home, P., Diabet. Med. 6: 559-560, 1989), and “themetabolic cardiovascular risk syndrome” (Hjermann, I., J. Cardiovasc.Pharmacol. 20: S5-S 10, 1992). The related complications or disorders ofMS-X include dyslipidemia (hypertriglyceridemia and low high-densitylipoprotein (HDL)-cholesterol), a prothrombotic state, type 2 diabetes(adult onset diabetes), insulin resistance/hyperinsulinemia,hypertension, and abdominal obesity. Grundy, S. M. Am J Cardiol. 81:18B-25B, 1998.

Although the patient may not have any symptoms from MS-X, the attendingphysician could identify the following as signs of the condition: (1)elevated insulin levels, due to insulin resistance; (2) type IIdiabetes; (3) central obesity (a disproportionate amount of body fat inthe abdominal region); (4) hyperlipidemia (high levels of fats (lipids)in the blood, which include LDL (“bad”) cholesterol and triglycerides.In addition, the size of the LDLs may be smaller than usual, which ismore likely to promote atherosclerosis); (5) low level of HDL (“good”)cholesterol; (6) hypertension (high blood pressure); (7) elevated levelsof blood factors that promote blood clotting, such as plasminogenactivator inhibitor-1 (PAI-1) and fibrinogen; (8) hyperuricemia (highlevels of uric acid in the blood); and (9) microalbuminuria (smallamounts of the protein albumin, found on urine tests). Grundy S. M., Am.J. Cardiol. 83: 25F-29F, 1999.

Independently, each of these complications or disorders promotesatherosclerosis. However, when grouped together, they are increasinglyatherogenic and enhance the risk of cardiovascular disease (CVD) at anylow density lipoprotein cholesterol level. In addition to increasing apatient's risk of CVD, MS-X may enhance the development of stroke, type2 diabetes (Lebovitz, H. E., Exp. Clin. Endocrinol. Diabetes 109:S135-S148, 2001), diabetic nephropathy, retinopathy, and distalneuropathy (Isomaa, B. et al., Diabetologia 44:1148-1154, 2001).

Using the above-mentioned features, one estimate suggests that as manyas 50 to 75 million people in the United States may exhibit significantsigns of MS-X by 2010. Hansen, B. C., Ann. N Y Acad. Sci. 892: 1-24,1999.

According to the World Health Organization (WHO) guideline, anindividual is diagnosed to have MS-X if the features are present: a)hypertension (>140 mm Hg systolic or >90 mm Hg diastolic); (b)dyslipidemia, defined as elevated plasma triglycerides (150 mg/dL)and/or low high-density lipoprotein (HDL) cholesterol (<35 mg/dL in men,<39 mg/dL in women) concentrations; 3) obesity, defined as a high bodymass index (BMI) (30 kg/m²) and/or a high waist-to-hip ratio (>0.90 inmen, >0.85 in women); and 4) microalbuminuria (urinary albumin excretionrate≧20 μg/min). See WHO-International Society of HypertensionGuidelines for the Management of Hypertension. Guidelines Subcommittee.J. Hypertens. 17:151-183, 1999. By this standard, individuals with type2 diabetes must meet only 2 of the criteria in order to be diagnosedwith MS-X.

A similar guideline, established by the National Cholesterol EducationProgram (NCEP ATP III study) establishes that a person would have MS-X,if 3 or more of the following risk factors are present: (1) a waistcircumference>102 cm (40 in) for men or >88 cm (37 in) for women; (2) atriglyceride level≧150 mg/dL; (3) an HDL cholesterol level<40 mg/dL formen or <50 mg/dL for women; (4) blood pressure≧130/≧85 mm Hg; or (5) afasting glucose≧110 mg/dL. See JAMA 285: 2486-2497, 2001.

These guidelines assert that abdominal obesity rather than elevated BMIis more highly associated with MS-X and suggest that all patients withabdominal obesity should be evaluated for the possibility of thissyndrome. In addition, ATP III guideline has a lower diagnosticthreshold level than that of WHO for certain characteristics (i.e., HDLcholesterol and hypertension). Therefore, a higher proportion of thepopulation meets the ATP III standard for the diagnosis of MS-X.

The central features of MS-X are all highly related, entail numerousphysiological systems, and reveal a complex multifactorial etiology. Indyslipidemia, also known as the lipid triad, other lipid abnormalities,such as moderately raised (often high-normal) triglycerides, increasedpreponderance of small, dense LDL particles, and low levels of HDLcholesterol are included. Grundy, S. M. Circulation 95: 1-4, 1997.Dyslipidemia and insulin resistance are related metabolic conditions.Haffner, S. M. Am. J. Cardiol. 83: 17F-21F, 1999 and Ginsberg, H. N. andHuang, L. S., J. Cardiovasc. Risk 7: 325-331, 2000.

With respect to a prothrombotic state, insulin resistant patients oftenexperience changes in coagulation factors that may promote arterialthrombosis and inflammation. Grundy, S. M. et al., Circulation 100:1134-1146, 1999. A procoagulant state may increase the formation ofatherosclerotic plaques and the size of thrombi following the rupture ofplaques. Commonly identified conditions in the MS that are related to aprothrombotic state include activation of endothelial cells, promotionof LDL oxidation, enhanced platelet aggregation, activation of factorVII, increased levels of factor IX, factor X, and prothrombin, andincreased concentrations of PAI-1. Peroxisome proliferator-activatedreceptor-α (PPAR-α), a major regulator of intra- and extracellular lipidmetabolism, may play a role in atherogenic dyslipidemia andinflammation. Gervois, P. et al., Clin. Chem. Lab. Med. 38: 3-11, 2000.Activation of the PPAR-αligand-binding domain may assist fatty acidmetabolism in the liver by promoting transcription of certain targetgenes, such as fatty acid binding protein. In addition, PPARs may play acentral role in regulating the interaction between HDL cholesterol andapolipoprotein (apo) B-containing lipoproteins. Pineda, T. I., et al.,Curr. Opin. Lipidol. 10: 151-159, 1999.

Overproduction of insulin leads to hypertension. WHO guidelines suggestthat patients receiving anti-hypertensive treatment and/or havingelevated blood pressure (>140 mm Hg systolic or >90 mm Hg diastolic) areat risk for MS. J Hypertens. 17: 151-183, 1999. Hypertension has beenwell established as a metabolic disorder and is predictive of insulinresistance. Lind, L. and Lithell, H. Am. Heart J. 125: 1494-1497, 1993.As many as 50% of the anti-hypertensive patients have comorbid insulinresistance and hyperinsulinemia. McLaughlin, T. and Reaven, G.,Geriatrics 55: 28-35, 2000. The use of an appropriate pharmacologicagent to reduce blood pressure may lessen the signs of insulinresistance in patients who exhibit both conditions. Lowering elevatedblood pressure may also improve a patient's lipid profile. Weidmann, P.et al., Am. Heart J. 125: 1498-1513, 1993.

Increased blood pressure independently increases the risk ofatherosclerosis, presumably by promoting the entry of LDL into thesubendothelial space, and may exacerbate other metabolic abnormalities.Homstra, G. et al., Br. J. Nutr. 80: S113-S146, 1998.

For diagnosis of MS-X, a variety of blood test are used to measurelevels of glucose, insulin, triglycerides, cholesterol, uric acid,fibrinogen and PAI-1. In addition, blood pressure and body weight shouldbe measured and evaluated.

Currently, the only known treatment strategies that addresses all thefactor of MS-X are weight loss and exercise. Medications are given butphysicians would usually encourage the MS-X patients to change theirlife style such as decreasing the amount of fats and oils in their diet,avoiding concentrated sweets, quitting smoking and avoiding excessivealcohol use.

Besides the above-mentioned strategies, several groups have disclosedthe use of specific drugs to treat MS-X and its related complications.Below is a brief summary of their disclosures.

U.S. Pat. No. 6,166,049 discloses a method for the treatment orprophylaxis of syndrome X in a human or non-human mammal byadministering an effective, non-toxic and pharmaceutically effectiveamount of an agonist of peroxisome proliferator-activator receptor-γ and-α (PPAR-γ and PPAR-α). The inclusion of PPAR-α in a PPAR-γanti-hyperglycaemic agent will result in a reagent with enhancedtherapeutic potential in the syndrome X etiology due to an enhancedhypolipaedemic effect. The invention provides a prophetic examplerelating to the efficacy of the compounds on blood glucose and plasmalipids in a genetically diabetic mouse.

U.S. Pat. No. 6,197,765 discloses a treatment for MS-X and its relatedcomplications, including diabetes complications, by administering a doseof diazoxide to inhibit the release of insulin and proinsulin, lowerweight, reduce levels of circulating cholesterol and triglycerides,lower blood pressure and prevent and reverse diabetic complications.

U.S. Pat. No. 6,410,339 discloses the use of synthetic cortisol agoniststhat have glucocorticoidal and/or mineral corticoidal effects, e.g.,dexamethasone, for preparing a system to diagnose MS and its relatedconditions such as belly fatness, insulin resistance including risk ofdeveloping senile diabetes, i.e., diabetes type II, high blood fats andhigh blood pressure. The dose of cortisol agonist is in an intervalwhere a difference is obtained in the inhibitory effect of theautoproduction of cortisol in individuals suffering from MS, compared tonormal values.

U.S. application serial No. 20020165237 by Fryburg et al. teaches theuse of selective cyclic guanosine monophosphate (cGMP) specificphosphodiesterase type 5 inhibitors, such as sildenafil, for thetreatment of insulin resistance syndrome (IRS). Sildenafil has beenshown to be effective in the treatment of male erectile dysfunction.Sildenafil increases the intracellular concentrations of nitric oxide(NO)-derived CGMP. This accumulation would amplify the vasodilatory,metabolic, and anti-atherogenic effects of the available nitric oxideand insulin. According to the inventors, such treatment may lead toclinically relevant improvements in blood pressure and/or blood sugarand/or lipids and/or uric acid, and/or procoagulant factors. Thistreatment can occur alone or in combination with other therapeutics thatimprove IRS which, in turn, should reduce the risk of the development ofcardiovascular disease in some patients, as well as other complicationsof individual disorders (including, but not limited to diabeticneuropathy, nephropathy, and retinopathy).

U.S. application serial No. 20020037861 A1, by Plata-Salaman et al.,discloses the use of anticonvulsant derivatives in preventing thedevelopment of type II diabetes mellitus and syndrome X. One of theanti-convulsant derivatives, 2,3:4,5-bis-O-(1-methylethylidene)-β-D-fructopyranose sulfamate (known astopiramate), has been demonstrated in clinical trials of human epilepsyto be effective as adjunctive therapy or as monotherapy in treatingsimple and complex partial seizures and secondarily generalizedseizures. Using a homozygous diabetic mouse model (ob/ob), treatmentwith topiramate resulted to a significantly lower levels of bloodglucose, triglycerides, and insulin and glycosylated hemoglobin than incontrol ob/ob mice not given topiramate. According to the inventors,these findings demonstrate that topiramate can reduce or preventpathophysiological signs associated with syndrome X. In addition, theamelioration of diabetic condition by topiramate is not dependent on areduction in body weight.

Although drug therapy has proven to be effective in reducing andtreating insulin resistance, adult onset diabetes, and MS-X and itsrelated complications, it leads to side effects, such as weight gain,water retention, slight risk of cardiac events and hypoglycemia,gastrointestinal problems including nausea, flatulence, and diarrhea,lactic acidosis, reduced absorption of vitamin B12 and folic acid, andreduced iron absorption.

Accordingly, there is a need to provide an improved method of treatingthe above-mentioned conditions that avoids or minimizes these sideeffects. There is a continuing need to provide an early method fortreating these conditions to avoid the emergence of costly and disablingdegenerative conditions, as described above.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that treatment ofmammalian subjects with intravenously administered liposomes of the typedescribed herein results in a reduction of insulin resistance andimprovement of symptoms of adult onset diabetes, and metabolic syndromeX and its related complications, with negligible or no side effects.

In one general embodiment, the method is used to treat a mammaliansubject having the above-mentioned conditions, as evidenced by thereduction of one or more of blood glucose, insulin, total cholesterol,LDL cholesterol, triglyceride, creatine kinase (CK), creatine kinase-MB(CK-MB), Hb-A1_(c), lipoprotein (a), SGOT and SGPT of the mammaliansubject. In a preferred embodiment, the method comprises intravenouslyadministering to a mammalian subject a therapeutically effective amountof liposomal suspension of lipoprotein small unilamellar vesicles (SUVs)comprising predominantly phospholipids.

In one aspect, the liposomal suspension of lipoprotein SUVs comprisephospholipids selected from the group consisting of phosphatidylcholine,phosphatidylglycerol and phosphatidylserine. In another aspect, thephosphatidylcholine is 1-palmitoyl, 2-oleoyl phosphatidylcholine and1-palmitoyl, 2-linoleoyl phosphatidylcholine and can be derived fromeggs.

In another embodiment, the lipoprotein SUVs comprise predominantly ofphosphatidylcholine having a transition temperature of less than about37° C., preferably about −10 to 24° C. The liposomal suspension oflipoprotein SUVs further comprises sphingomyelin, cholesterol or othersterols, in an amount less than about 40 mole percent. In yet anotherembodiment, the lipoprotein SUVs are empty.

In one embodiment, the liposomal suspension is administered one to threetimes per week to a mammalian subject having the above-describedconditions at a dose of about 50 mg-1 g total lipid/kg body weight,preferably at a dose of about 200-450 mg total lipid/kg body weight. Theadministration can be achieved via intravenous injection or intravenousinfusion.

The features and details of the invention will become more apparent andappreciated by one skilled in the art to which this invention pertainsfrom the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a” or “an” means “one or more.”

“Mammalian subject” refers to both a veterinary animal or a human.

“Glycosylated or glycated haemoglobins (Hb-A_(1c))” refers to normalhaemoglobin to which a glucose molecule becomes added in a non-enzymaticmanner. The percentage haemoglobin that is glycated is directlyproportional to the time that red blood cells have been exposed toglucose, and to glucose concentrations. Measurement of the glycatedhaemoglobin fraction gives an integrated picture of the average bloodglucose concentration during the half life of the cells—that is over thelast 60 days. Hb-A_(1c) is usually given as a percentage of the totalhemoglobin. Hb-A_(1c) levels in individuals with normal glucosetolerance have a normal range of 4.3% to 6.3% (Peters, A. L. et al. J.Am. Med. Assoc. 276:1246-52, 1996).

“Serum Glutamic-Oxaloacetic Transaminase” (“SGOT”) or AspartateAminotransferase” (“AST”) is an enzyme found primarily in the liver,heart, kidney, pancreas, and muscles. It is elevated in patients withmuscular disease, myocardial injury and renal infarction. Decreasedlevels can be found in Vitamin B deficiency and pregnancy. The normalvalue range is 0-50 U/1. The optimal adult reading is 21 U/l.

“Serum Glutamic-Pyruvic Transaminase” (“SGPT”) or AlanineAminotransferase” (“ALT”) is an enzyme found primarily in the liver butalso in the heart and other tissues. Decreased SGPT in combination withincreased cholesterol levels is seen in congested liver cases. Increasedlevels are seen in mononucleosis, alcoholism, liver damage, kidneyinfection, chemical pollutants or myocardial infarction. The normalvalue range is 0-41 U/l. The optimal adult reading is 24 U/l.

“C-Reactive Protein” (“CRP”) is a protein present in many acuteinflammatory conditions and is a significant risk factor incardiovascular disease. Higher levels of CRP may play a role inabdominal obesity and the onset of type II diabetes or AOD. The normalrange for CRP is 0-0.5 mg/dl.

“Creatine Kinase” (“CK”) is an enzyme found in the heart, brain, andskeletal muscle. Blood levels of CK rise when the muscles or heart cellsare injured. CK occurs in three major forms, called isoenzymes, namely:(1) CK-MB (found mostly in the heart muscle); (2) CK-BB (found mostly inthe brain); and (3) CK-MM (found in the heart and other muscles). CK-MBlevels, along with total CK, are tested in persons who have chest painto determine whether they have had a heart attack. Since a total CKwould indicate damage to either a heart or other muscles, a high CK-MBsuggests that the damage was to the heart muscle. The normal ranges forCK and CK-MB are 0-195 U/l and 0-24 U/l, respectively.

“Glucose intolerance” refers to the inability of the body to utilizeglucose in blood circulation. Glucose intolerance is increased indiabetes mellitus and in endocrine disorders such as polycystic ovarysyndrome.

“Insulin” refers to the hormone that helps shuttle glucose from theblood to the cells. The beta cells in areas of the pancreas called theislets of Langerhans usually make insulin.

“Insulin sensitivity” (or “insulin receptivity”) is the normal andpreferable state, in which the body's cells remain sensitive (orreceptive) and responsive, to insulin action.

“Insulin resistance” refers to a state wherein abnormally high glucoselevels trigger an increase in insulin to remove this sugar from thebloodstream. Often, the body's cells start to ignore high insulin levelsand therefore become resistant to the hormone's effects. Insulinresistance allows glucose levels to rise and stay high. However, it doesnot always lead to diabetes.

Hypertension is defined as elevated blood pressure.

Dyslipidemia is defined as elevated plasma triglycerides and/or lowhigh-density lipoprotein (HDL) cholesterol concentrations.

Obesity is defined as a high body mass index (BMI) and/or a highwaist-to-hip ratio.

“Lipid replacement therapy” refers to a therapy in which the treatmentmodifies the composition of the cell membranes in both tissue culturesas well as the whole organism particularly, but not limited to, the redblood cells, blood vessels, heart cells and kidney cells in such a wayas to cause the cholesterol to phospholipid mole ratio as well as thesphingomyelin to phosphotidylcholine mole ratio to decrease and revertto values found in young cells or tissues of young organisms.

“Empty” liposomes refers to liposomes that do not contain entrapped orencapsulated drug.

“Small unilammelar vesicles (SUVs)” refer to small single-bilayerliposomes having particle sizes ranging predominantly between 20 and 120nm. The SUVs of the present invention can be empty. They comprisephospholipids, preferably phospholipids selected from the groupconsisting of phosphatidylcholine, phosphatidylglycerol, andphosphatidylserine. The phosphatidylcholine may be an eggphosphatidylcholine.

The phrase “milligram or gram total lipid/kg body weight” refers to theamount of total lipids in milligrams or grams comprising the lipoproteinSUVs per kilogram body weight. Total lipids may includephosphatidylcholine, sphingomyelin, cholesterol, phosphatidylserine,phosphatidyglycerol or any other lipids described in the presentinvention.

A “significant improvement” in a disease state is a measurable degree ofimprovement, as indicated by either a clinical or biochemical indicator,in the disease state. Typically, a significant improvement in a diseasestate is one which results in an improvement of a parameter with a knowncorrelation to the disease state of at least five percent.

The terms “elevated blood glucose, insulin, total cholesterol, LDLcholesterol, and triglyceride”, as used herein, refer to concentrationsof blood glucose, insulin, total cholesterol, LDL cholesterol, andtriglyceride that are on average elevated above the normal averageconcentrations when measured at various times over the course of a week.A normal range for glucose is generally between 55-115 mg/dl; fortriglyceride, 0-200 mg/dl; for total cholesterol, 100-200 mg/dl; and forLDL cholesterol, 0-155 mg/dl.

“Oral glucose tolerance test (OGTT)” refers to a two to three-hr glucosetolerance test that measures blood glucose levels four to five timesover a 2-3-hour period. The patient is administered an oral dose ofglucose solution (75 to 100 grams of an extremely sweet drink), whichshould cause glucose levels to rise in the first hour, and then fallback to normal within two to three hours as the body produces insulin tonormalize glucose levels. This test us used to confirm a diagnosis ofdiabetes mellitus or gestational diabetes (and to diagnose othermetabolic diseases).

OGTT is a more sensitive test than the fasting plasma glucose test, andinvolves multiple blood draws to monitor insulin production, it canoften detect cases of mild diabetes that may be missed by the fastingtest. The most commonly used test protocols include the Wilkerson pointsystem; the Fajans-Conn system, or the National Institutes of Health(NIH) system. On average, normal glucose levels typically peak at160-180 mg/dl from 30 minutes to 1 hour after administration of the oralglucose dose, and should then return to fasting levels of 140 mg/dl orless within a 2 to 3 hour period. Factors such as age, weight, and racecan influence results, as can recent illnesses and certain medications.For example, older individuals will have an upper limit increase of 1mg/dl in glucose tolerance for every year over age 50. Glucose levelsthat quickly rise above normal levels (i.e., 200 mg/dl or higher) andtake longer to normalize usually indicate diabetes mellitus.

Insulin resistance should be diagnosed by measuring insulinlevels—fasting levels alone, or with a glucose tolerance test plusinsulin (sometimes called an IGTT). Diabetes may be diagnosed based onblood glucose levels alone.

Oral Glucose Tolerance Test—Glucose and Insulin Values

Normal Normal Time Glucose Insulin (hour) Values Values Interpretationof Results Fasting <126 mg/dl <10 mIU/ml Normal glucose results are70-90, 111 or over is impaired, 126 or over is diabetic. Insulin levelsabove 10 show insulin resistance.   0.5 <200 mg/dl 40-70 mIU/ml A trulynormal glucose response will not exceed 150. 1 <200 mg/dl 50-90 mIU/mlSome want to lower the threshold on glucose to < 180 to identify earlystages of diabetes. Insulin > 80 shows insulin resistance, or a level 5times that of the fasting level (i.e., a fasting of 11 followed by a 1hour > 55) 2 <140 mg/dl 6-50 mIU/ml A truly normal glucose response is110 or lower. Insulin > 60 is IR. 3 <120 mg/dl 4 <120 mg/dl

While not wishing to be bound by a particular theory, the presentinventor discovered that a liposomal suspension known to be effective intreating conditions associated with aging, such as heart disease, isalso useful in treating insulin resistance, adult onset diabetes andmetabolic syndrome-X and its related complications. The treatment uses asimilar protocol to that which is known in the art. However, thistreatment must be adapted to the treatment of insulin resistance, adultonset diabetes and metabolic syndrome X and its related complications,as would be apparent to the ordinary skilled physician.

II. Preparation of Liposomal Composition

The present invention involves intravenous administration of atherapeutic liposomal suspension to a subject having insulin resistance,adult onset diabetes, and metabolic syndrome X and its relatedcomplications. The liposomal suspension comprises lipoprotein smallunilammelar vesicles (SUVs), comprising predominantly phospholipidsselected from the group consisting of phosphatidylcholine,phosphatidylglycerol, and phosphatidylserine. The phosphatidylcholinemay be an egg phosphatidylcholine. Preparation of the lipoprotein SUVsof the present invention is illustrated in the Examples and in thesections which follow.

A. Preparation of Liposomes: Composition

Lipoproteins are high molecular weight particles that are primarilyresponsible for lipid transport, namely of triglycerides and cholesterolin the form of cholesteryl esters, through the plasma. Five majorclasses of naturally-occurring lipoproteins are known to circulate inplasma, each differing in lipid composition, apoprotein composition,density, size, and electrophoretic mobility.

Each lipoprotein particle is composed of a non-polar core region, asurrounding phospholipid surface coating containing small amounts ofcholesterol, and exposed at the surface. In vivo, the lipoproteinparticles can become associated with apoproteins, e.g., apoproteins Aand C, that are responsible for binding to receptors on cell membranesand directing the lipoprotein carrier to its intended site ofmetabolism.

In one preferred embodiment, described and used in the examples below,the lipoprotein SUVs comprise predominantly (more than 50 mole percent,preferably more than 80-90 mole percent) of phosphatidylcholine (PC)having a phase transition temperature less than about 37° C., preferablyabout −10 to 24° C., e.g., below about 5° C.

PC phospholipids include those phospholipids having a choline moiety andwhere the fatty acid chain portion of the phospholipid may vary inlength and degree of unsaturation. In addition, PC phospholipids alsoinclude synthetic PCs that are not crystalline at body temperature(e.g., those containing at least one double bond) yet are resistant tooxidation (e.g., those that do not have double bands, such as1-palmitoyl, 2-oleoyl PC(POPC)). PC phospholipids may further includenatural or synthetic phospholipids, alone or in mixtures havingsupplemented or replaced hydrophobic or amphipathic material that stillmaintains a liposomal or micellar structure.

One preferred vesicle composition includes egg PC, which has atransition temperature of −5° C., that contains predominantly1-palmitoyl, 2-oleoyl PC and 1-palmitoyl, 2-linoleoyl PC. Alternatively,phosphatidylcholine may be isolated from rat liver (Newman, H. A. I. etal., J. Lipid Res. (1961) 2:403-11), followed by purification on alumina(Shinitzky, M. et al., J. Biol. Chem. (1974) 249:2652).

The lipoprotein SUVs may be composed entirely of egg PC, or may containother lipid components which (i) are not immunogenic, (ii) do notcontribute a significant portion, i.e., more than 25-50 mole percent, oflipids with high phase transition temperature. Additional components mayinclude negatively charged lipids, such as phosphatidylglycerol (PG) orphosphatidylserine (PS). Addition of PG would make the SUVs negativelycharged or charge other components of the lipoprotein SUVs to preventaggregation during storage. If the lipoprotein SUVs is composed entirelyof PC, the mole percentage of PG and PS is less than 1% with respect toPC. However, if PC is not a major component of the lipoprotein SUVs, themole percentage of PG and PS would be more than 1% with respect to PC.The lipoprotein SUVs may also encompass sphingomyelin (SM), cholesterolor other sterols, in an amount preferably less than about 40 molepercent. Other components may also include diacylglycerol,phosphatidylinositol, oxidized lipids, lysophosphatidylcholine, andproteins, such as phospholipid transfer proteins (PLTP; see BiomembranesStructural and Functional Aspects, M. Shinitzky (ed.), 1994, at page 40)and amniophospholipid translocase (either as a 116-kd Mg²⁺ ATPase(Morot, G. et al., Biochemistry 28: 3456, 1989; Morot, G. et al., FEBSLett. 266: 29, 1990) or as a 32-kd protein (Schroit, A. J. et al.,Biochim. Biophys. Acta 1071: 313, 1991)).

Lipid protective agents, such as α-tocopherol, α-tocopherol acetate, orα-tocopherol succinate, may also be included in the lipids forming thelipoprotein SUVs, to protect the lipid components against free radicaldamage. Typically such agents are included at a mole percentage betweenabout 0.5% and 2%. It may be advantageous to add α-tocopherol to thelipoprotein SUVs to maintain a balance between vitamin E andpolyunsaturated lipids in the lipoprotein SUVs. Alternatively, thelipoprotein SUVs can be prepared and stored in an inert gas atmosphere,e.g., nitrogen, argon and the like.

B. Preparation of Unsized Liposomes

A variety of methods for producing lipoprotein SUVs are available, andthese have been extensively reviewed (Szoka, F. et al., Ann. Rev.Biophys. Bioeng. (1980) 9:467). In general, these methods producelipoprotein SUVs with heterogeneous sizes from about 0.02 to 10 micronsor greater. As will be discussed below, lipoprotein SUVs which arerelatively small and well-defined in size are preferred for use in thepresent invention, hence a second processing step for reducing the sizeand size heterogeneity of liposomal suspensions will usually berequired.

In one preferred method for forming the initial liposome suspension asdescribed in Example 1, the vesicle-forming lipids are taken up in asuitable organic solvent system, preferably in a siliconized glassvessel, and dried in vacuo or under an inert gas to form a lipid film.An aqueous suspension medium, such as a sterile saline solution, isadded to the film, and the vessel is agitated (e.g., on a shaker orusing a sonicator) until the lipids have hydrated to completion,typically within about 1-2 hours. The amount of aqueous medium added issufficient to produce a final liposome suspension containing preferablybetween about 5 and 30 g total lipid per 100 ml media, preferably 10 gtotal lipid per 100 ml media.

During the hydration stage, the lipids hydrate to form multilamellarvesicles (MLVs) with sizes ranging between about 0.5 microns to about 10microns or larger. In general, the size distribution of MLVs can beshifted toward slightly smaller sizes by hydrating the lipids under morevigorous agitation conditions.

The aqueous medium used in forming the lipoprotein SUVs may containwater-soluble agent(s) which enhance the stability of the liposomes uponstorage. A preferred stabilizing agent is an iron-specifictrihydroxamine chelating agent, such as desferrioxamine. The use of thiscompound in reducing lipid peroxidation and free radical damage indrug-containing liposomes has been reported in U.S. Pat. No. 4,797,285.Briefly, it was shown that the combination of a lipophilic free-radicalquencher, such as α-tocopherol, and the water-soluble chelator gavesubstantially better protection against lipid peroxidation damage thandid either of the protective agents alone. The chelator is included inthe aqueous medium in molar excess of the amount of free iron in themedium. Typically, a chelator concentration of between about 10-200micromolar is sufficient for reducing lipid peroxidation and freeradical damage.

C. Sizing Liposomes: SUV Preparation

The suspension of lipoprotein SUVs prepared as described above ispreferably further treated to produce liposomes having a desired sizeand size homogeneity.

The liposome suspension is generally sized to achieve a selective sizedistribution of vesicles in a size range less than about 1.2 micron andpreferably less than about 0.8 microns. Liposomes in this size range canbe readily sterilized by filtration through a depth filter. Smallervesicles also show less tendency to aggregate on storage, thus reducingthe potential for serious vascular blockage problems upon intravenousadministration of the final liposomal composition of the presentinvention. Finally, lipoprotein SUVs which have been sized down to thesubmicron range possess more uniform biodistribution and drug clearancecharacteristics.

Preferred lipoprotein SUVs, i.e., single-bilayer liposomes, have sizesbetween about 0.02 to 0.12 microns. SUVs have been shown to possessrelatively long blood circulation half lives, when administeredintravenously, as described in U.S. patent application No. 6,235,308,filed Jun. 10, 1994. Briefly, as described therein, plots of liposomeretention in the bloodstream, measured up to 1,000 minutes after IVinjection, revealed that significant quantities of liposomes remained inthe bloodstream even at 1,000 minutes.

Several techniques are available for reducing the sizes and sizeheterogeneity of liposomes, in a manner suitable for preparing thelipoprotein SUVs of the present invention. Ultrasonic irradiation of aliposome suspension either by bath or probe sonication produces aprogressive size reduction down to SUVs.

Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, MLVs are recirculated through a standard emulsion homogenizeruntil selected liposome sizes, typically less than 0.1 microns, areobserved.

Extrusion of liposomes through a small-pore polycarbonate membrane is aneffective method of reducing liposome size down to a relativelywell-defined size distribution. An average range is between about 0.03and 1 micron, depending on the pore size of the membrane, such asdescribed in Example 2. Typically, the suspension is cycled through themembrane several times until the desired liposome size distribution isachieved. The lipoprotein SUVs may be extruded through successivelysmaller pore membranes, to achieve a gradual reduction in liposome size.

Liposome particle sizes can be determined by a number of techniquesincluding electron microscopy, comparative chromatography (Bisgaier, C.L. et al., J. Biol. Chem. (1989) 264(2):862-866) and quasi-elastic lightscattering.

The size-processed liposome suspension may be readily sterilized bypassage through a sterilizing membrane having a particle discriminationsize of about 0.2μ, such as a conventional 0.22μ depth membrane filter.If desired, the liposome suspension can be lyophilized for storage andreconstituted shortly before use.

III. Methods of Treating Insulin Resistance, Adult Onset DiabetesDisease, and Metabolic Syndrome X and its Related Complications

This section describes treatment methods which involve intravenousadministration of the liposomal suspension described above. In all ofthese methods, the suspension is administered intravenously at a doseand dosing frequency effective to produce a desired improvement in thetreated condition.

A preferred dosing frequency is one, two or three times per week. Thedosing periods, e.g., two weeks, may be interrupted by a wash-outperiod, typically of 1-4 weeks. The treatment, e.g., involving repeatingdosing and wash-out periods, may continue over an extended period ofseveral months or more.

In a preferred embodiment, the liposome suspension is administered oneto three times per week, at a dose of about 50 mg-1 g total lipid/kgbody weight per dose, preferably between about 200-450 mg total lipid/kgbody weight per dose. Administration may be by i.v. (intravenous)injection, or i.v. drip (infusion). The lipoprotein SUVs may besuspended in sterile saline or in a nutritional or drug-containingbuffer or medium, such as a glucose/salt medium, to combine liposometreatment with other parenteral therapy.

Administration of the liposomal suspension is continued until asignificant and measurable improvement of the disease is observed andwherein the levels of one or more of blood glucose, insulin, totalcholesterol, LDL cholesterol, triglyceride, creatine kinase (CK),creatine kinase-MB (CK-MB), Hb-A1_(c), lipoprotein (a), SGOT and SGPTfall back within the normal range or are significantly reduced.

A liposomal suspension is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. It is physiologically significant if its presence resultsin a detectable change in the physiology of a recipient subject. Inparticular, an amount of a liposomal suspension administered accordingto the present invention is physiologically significant if it results ina reduction of the levels of one or more of blood glucose, insulin,total cholesterol, LDL cholesterol, triglyceride, creatine kinase (CK),creatine kinase-MB (CK-MB), Hb-A1_(c), lipoprotein (a), SGOT and SGPT.

EXAMPLES

The following examples illustrate various methods for preparing liposomecompositions and using the compositions in the treatment method of theinvention. The examples are intended to illustrate, but in no way limit,the scope of the invention.

Materials

Egg phosphatidylcholine (egg PC) may be purchased from Avanti PolarLipids (Alabaster, Ala.) or Lipoid KG (Ludwigshafen, Germany). The eggPC was determined to be greater than 99% pure. The egg PC fatty acidcomposition was similar to the reported composition (Hertz, R. et al.,Chem. Phys. Lipid (1975) 15:138). The main PCs of the preparationincluded 1-palmitoyl, 2-oleoyl PC and 1-palmitoyl, 2-linoleoyl PC.

Example 1 Preparation of Small Unilamellar Vesicles by Sonication

Egg PC dissolved in chloroform was placed in a 100 ml vessel and driedto a thin film under an inert atmosphere of nitrogen. Sterile saline wasadded to the lipid film to a final concentration of about 100 mg/ml, andthe lipid film was hydrated with swirling. The resulting multilamellarvesicle (MLV) suspension was then bath sonicated for 1 hour using a HeatSystem Sonicator, Model 375W, at a power setting of 40-50% full value.The temperature of the suspension was maintained at about 4° C. duringsonication. Large vesicles or MLVs were separated from the sonicatedsuspension by ultracentrifugation at 100,000 g for 1 hour (Barenholz, Y.et al., Biochemistry (1977) 16:2806). The remaining suspension of SUVs,having a concentration of about 100 mg/ml, was then filter sterilized.

Example 2 Preparation of Small Unilamellar Vesicles by Extrusion

Homogeneous small unilamellar vesicles (SUVs) of egg PC for human usewith an average diameter of 65 nm±10 nm in size, in 0.15M NaCl, wereprepared by extrusion using serial filtration through polycarbonatefilters in a GH 76-400 pressure cell (Nucleopore) (Anselem, S., et al.In Gregoriadis, G. (ed). LIPOSOME TECHNOLOGY, pp. 501-524, CRC Press,Boca Raton, Fla. (1993)). These vesicles were empty SUVs.

Liposomal particle size was measured by Nicomp submicron laser particlesizer, by Quasielectric light scattering or comparable method. It canalso be determined using a Coulter model N4 sub-micron particle analyzerequipped with a size distribution processor analyzer (Barenholz et al.In Gregoriadis, G. (ed), LIPOSOME TECHNOLOGY, pp. 524-607, CRC Press,Boca Raton, Fla. (1993)). The final extrusion step was through a 0.05micrometer pore polycarbonate filter. Egg PC SUV's should be sterile andpyrogen-free and were sterilized by filtration through sterile 0.22micrometer Millipore filters. They were packaged in 100 ml transparentmoulded hydrolytic class I bottles, evacuated with nitrogen (N) withteflon coated standard stoppers (20 mm), and sealed with an Alu-cap withPE-disc.

The final product had a more than 99% purity. Total impurity values wereNMT 1%, as measured by HPLC, GC/FD or comparable procedure. There was nosingle impurity greater than 0.3%. Total oxidation values were NMT 1%,as measured by UV/VIS spectrometry at 234, 268 and 278 nm.

Below is some additional information relating to the final SUV liposomalproduct:

Lipid concentration: 5-20%, preferably 10%.

Chemical stability: Hydrolysis and peroxidation<1%.

Sterility: Passes FDA mandated standards for sterile solution. NMT 100CFU per test sample. Conforms to USP standards.

Storage Conditions: Room Temperature Refrigerated TemperatureAccelerated Temperature (25° C., 60% RH) (4° C., 45% RH) (40° C., 75%RH) 1 week 1 week 1 week 2 weeks 1 month 1 month 1 month 2 months 2months 2 months 3 months 3 months 3 months 6 months 6 months 6 months

Dose regimen: 50 mg-1 g total lipid/kg body weight.

Dosage form: White-yellowish translucent dispersion for intravenousinfusion.

Contraindication: Allergy to eggs; having hemolytic red blood cells.

Adverse effects: No adverse effects found.

Liposomal Concentration: 100 mg/ml

Fatty acid composition of the egg PC (by weight): Palmitic acid 35.6%Stearic acid 12.6% Oleic Acid 27.8% Linoleic Acid 17.9% Arachidonic Acid 9.5% Docosapentaenoic acid  0.7% Docosahexaenoic acid  2.0%

Complete Liposomal Suspension: 10 g phospholipids of which: 9.9 g eggPC, 0.2-0.50 g cholesterol and 0.05-2.0 g sphingomyelin 0.012 gDesferal ® 0.9 g sodium chloride

Sterile pyrogen-free water for injection to 100 ml.

Example 3 Alternative Preparation of Small Unilamellar Vesicles byExtrusion

Homogeneous small unilamellar vesicles (SUVs) of egg PC for human usewith an average diameter of 60 nm±5 nm in size, were prepared byextrusion using filtration through polycarbonate membrane filters usingan Aviston EmulsiFlex-C50 homogenizer with Supor Cap™ and SuporDCF™serial layer disposable filters (220 nm, 180 nm and 80 nm). Thesevesicles were empty SUVs.

Liposomal particle size was measured by Solvias AG, Basel, Switzerlandsubmicron laser particle sizer, by Quasielectric light scattering orcomparable method. It can also be determined using a Coulter model N4sub-micron particle analyzer equipped with a size distribution processoranalyzer (Barenholz et al. In Gregoriadis, G. (ed), LIPOSOME TECHNOLOGY,pp. 524-607, CRC Press, Boca Raton, Fla. (1993)). The final extrusionstep was through a 0.08 μm pore polycarbonate membrane filter. Egg PCSUv's should be sterile, endotoxin (LAL)-free and pyrogen-free and weresterilized by filtration through sterile 0.22 μm pore polycarbonatemembrane filters. They were packaged in 100 ml transparent mouldedhydrolytic class II bottles, evacuated with nitrogen (N) with tefloncoated standard stoppers (20 mm), and sealed with an Alu-cap withPE-disc.

The final product had a more than 99% purity. Total impurity values wereNMT 1%, as measured by HPLC, GC/FID or comparable procedure. There wasno single impurity greater than 0.3%. Total oxidation values were NMT1%, as measured by UV/VIS spectrometry at 215, 233, and 279 nm.

Below are some additional information relating to the final SUVliposomal product:

Lipid concentration: 5-30%.

Chemical stability: Hydrolysis and peroxidation<1%.

Dosage form: White-yellowish translucent dispersion for intravenousinfusion.

Sterility: Passes FDA mandated standards for sterile solution. NMT 100CFU per test sample. Conforms to USP standards.

Storage Conditions: Refrigerated Temperature (4° C., 45% RH) 1 month 2months 3 months 5 months 6 months 7 months

Dose regimen: 50 mg-1 g total lipid/kg body weight.

Contraindication: allergy to eggs; having hemolytic red blood cells.

Adverse effects: No adverse effects found.

Fatty acid composition of the egg PC (by weight): Palmitic acid 35.6%Stearic acid 12.6% Oleic Acid 27.8% Linoleic Acid 17.9% Arachidonic Acid 9.5% Docosapentaenoic acid  0.7% Docosahexaenoic acid  2.0%

Complete Liposomal Suspension:

10 g phospholipids of which 9.0 g Egg PC (Lipoid) 0.2-0.5 g cholesteroland 0.05-2.0 g sphingomyelin 0.018 g Desferal ® 0.765 g sodium chloride(Merck) 0.279 g L-histidine (Fluka)

Adjust pH to 6.5 with 1 M sodium hydroxide or 1 M hydrochloric acid88.9945 g Sterile pyrogen-free water (final volume: 100 ml), andnitrogen q.s.

Example 4 Effects of Liposomal Treatment on an Insulin Resistant andAdult Onset Diabetic Patient

Patient 1 is a sixty-two year old woman having a body size of 160 cm anda body weight of 45 kg. She has a preknown dysregulation of lipidmetabolism and was diagnosed as an insulin-resistant metabolic patientfor 17 years. For treatment, she was intravenously infused (20-50drips/min) with two volumes of 90-ml per infusion (400 mg total lipid/kgbody weight) of the complete liposomal suspension, prepared according tothe procedure described in Example 3, six times within a 30 day period.

Patient 1 tolerated the liposomal therapy very well. She showed no signsor symptoms of any unwanted side effects. Even after several weeks oftherapy, she did not experienced any subjective or clinical adversesymptoms. Her laboratory results showed elevated total cholesterol buther LDL and cholesterol levels decreased while her lipoprotein (a) levelremained within the normal limits.

Before treatment, her glycated hemoglobin (Hb-A1_(c)) levels showed aslight increase or possible indication of glucose metabolic disturbance.After treatment, this level falls within the normal range. TABLE 1Laboratory Results for Patient 1 Before and After Treatment: BeforeAfter Test Reference Range Treatment Treatment Triglyceride 0-200 mg/dl114 mg/dl 71 mg/dl Total Cholesterol 100-200 mg/dl  349 mg/dl 312 mg/dl LDL Cholesterol 0-155 mg/dl 219 mg/dl 206 mg/dl  HDL Cholesterol 35-55mg/dl 107 mg/dl 92 mg/dl Glucose 55-115 mg/dl   94 mg/dl 92 mg/dl CK0-195 U/I 102 U/I 84 U/l CK-MB 3.9 N/A N/A Hb-A1_(c) 4.4-6.1 5.6 5.8Lipoprotein (a) 0.3 0.11 g/L 0.11 g/L

Several weeks after the liposomal treatment, an Oral Glucose ToleranceTest was performed by application of an appropriate dosage of glucose.After two hours of glucose application, serologic glucose levels andinsulin secretion were determined. Results showed that both glucoselevel and insulin secretion increased and reached a normalized levelafter two hours of glucose application. By contrast, results obtainedfrom three years prior to the liposomal treatment revealed that bothglucose level and insulin secretion failed to drop back to thenormalized level after two hours of glucose application.

A comparison of Oral Glucose Tolerance Test before and after treatmentis shown below: TABLE 2 Oral Glucose Tolerance Test - Glucose andInsulin Values Five Years Three Years Reference Before Before AfterRange Treatment Treatment Treatment Glucose, Fasting 70-120 mg/dl  93 9696 Glucose, 30 min 70-180 mg/dl 229 116 213 Glucose, 60 min 70-160 mg/dl176 173 217 Glucose, 120 min 70-130 mg/dl 177 143 82 Insulin, Fasting 3-15 μU/ml N/A 3.2 3.6 Insulin, 30 min 10-74 μU/ml N/A 13.2 28.5Insulin, 60 min 30-71 μU/ml N/A 35.4 105.6 Insulin, 120 min 10-48 μU/mlN/A 84.4 29.0

Therefore, treatment has improved the insulin resistance in the patient.

Example 5 Effects of Liposomal Treatment on A Metabolic Syndrome-XPatient

Patient No. 2 is a 51-year old man who has hypertension and a knownmetabolic disorder of lipids. He is overweight and has body size of 175cm and a body weight of 90 kg. His initial values for total cholesteroland triglycerides were very high. However, his LDL-cholesterol andHDL-cholesterol were within normal limits. In addition, there was anincrease of the liver enzyme, SGPT.

For treatment, patient No. 2 was intravenously infused (20-50 drips/min)with three-volumes of 90-ml per infusion (300 mg total lipid/kg bodyweight) of the complete liposomal suspension, prepared according to theprocedure described in Example 3, six times within a period of 19 days.

During the treatment period, the patient's blood pressure, pulsefrequency and oxygen load appeared normal. There were no subjective orclinical symptoms of unwanted effects.

Laboratory values after treatment revealed a decrease in totalcholesterol and triglycerides, in addition to a partial normalization ofSGOT (see Table 3), wherein treatment begins on May 10, 2002. TABLE 3Laboratory Results for Patient No. 2 Before and After Treatment:Reference Range Apr. 22, 2002 May 10, 2002 May 17, 2002 Jun. 28, 2002Aug. 28, 2002 Dec. 14, 2002 Triglyceride 0-200 mg/dl 314 mg/dl 798 mg/dl614 mg/dl 342 mg/dl 332 mg/dl 195 mg/dl Total Cholesterol 100-200 mg/dl233 mg/dl 244 mg/dl 356 mg/dl 218 mg/dl 204 mg/dl 197 mg/dl LDLCholesterol 0-155 mg/dl 125 mg/dl 48 mg/dl 185 mg/dl 103 mg/dl 94 mg/dl108 mg/dl HDL Cholesterol 35-55 mg/dl 45 mg/dl 36 mg/dl 48 mg/dl 47mg/dl 44 mg/dl 50 mg/dl Glucose 55-115 mg/dl 101 mg/dl 85 mg/dl 105mg/dl 110 mg/dl 100 mg/dl 107 mg/dl CK 0-195 U/l 191 U/l 238 U/l 530 U/l298 U/l 191 U/l 185 U/l CK-MB 0-24 U/l 25 U/l 23 U/l CRP 0-0.5 mg/dl 0.2mg/dl 0.0 mg/dl 0.0 mg/dl 0.0 mg/dl SGOT 0-50 U/l 29.5 U/l 26.8 U/l 40.8U/l 29.6 U/l 23.9 U/l 27.2 g/l SGPT 0-41 U/l 50.7 U/l 46.6 U/l 62.6 U/l57.5 U/l 42.8 U/l 39.5 g/l Lipoprotein (a) <0.3 g/dl 0.688 g/l 0.5 g/l0.59 g/dl 0.46 g/l 0.42 g/l 0.56 g/l Fibrinogen 200-400 mg/dl 243 mg/dlHb-A1_(c) 4.4-6.1% 5.6%

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

1. A method of treating insulin resistance, adult onset diabetes andmetabolic syndrome X and its related complications in a mammaliansubject, comprising intravenously administering to said mammaliansubject a therapeutically effective amount of liposomal suspension oflipoprotein small unilamellar vesicles (SUV's), comprising predominantlyphospholipids.
 2. The method of claim 1, wherein said phospholipids areselected from the group consisting of phosphatidylcholine,phosphatidylglycerol and phosphatidylserine.
 3. The method of claim 2,wherein said phosphatidylcholine is egg phosphatidylcholine.
 4. Themethod of claim 2, wherein said phosphatidylcholine is 1-palmitoyl,2-oleoyl phosphatidylcholine, 1-palmitoyl, 2-linoleoylphosphatidylcholine or a mixture thereon.
 5. The method of claim 2,wherein said phosphatidylcholine has a transition temperature of lessthan about 37° C.
 6. The method of claim 5, wherein said transitiontemperature is in the range of about −10 to 24° C.
 7. The method ofclaim 1, wherein said lipoprotein SUVs further comprise sphingomyelin,cholesterol or other sterols, in an amount less than about 40 molepercent.
 8. The method of claim 1, wherein said lipoprotein SUVs areempty.
 9. The method of claim 1, wherein said liposomal suspension isadministered one to three times per week to said mammalian subject at adose for each administration of about 50 mg-1 g total lipid/kg bodyweight.
 10. The method of claim 9, wherein said dose is about 200-450 mgtotal lipid/kg body eight.
 11. The method of claim 1, wherein saidliposomal suspension is administered by intravenous injection orintravenous infusion.
 12. A liposomal suspension of lipoprotein smallunilamellar vesicles (SUVs), comprising predominantly phospholipids foruse in the preparation of a medicament for treating insulin resistance,adult onset diabetes and metabolic syndrom X and its relatedcomplications in a mammalian subject.
 13. The use of claim 12, whereinsaid phospholipids are selected from the group consisting ofphosphatidylcholine, phosphatidylglycerol and phosphatidylserine. 14.The use of claim 13, wherein said phosphatidylcholine is eggphosphatidylcholine.
 15. The use of claim 13, wherein saidphosphatidylcholine is 1-palmitoyl, 2-oleoyl phosphatidylcholine,1-palmitoyl, 2-linoleoyl phosphatidylcholine or a mixture thereof. 16.The use of claim 13, wherein said phosphatidylcholine has a transitiontemperature of less than about 37° C.
 17. The use of claim 16, whereinsaid transition temperature is in the range of about −10 to 24° C. 18.The use of claim 12, wherein said lipoprotein SUVs further comprisesphingomyelin, cholesterol or other sterols, in an amount less thanabout 40 mole percent.
 19. The use of claim 12, wherein said lipoproteinSUVs are empty.
 20. The use of claim 12, wherein said medicament issuitable for administration one to three tim4es per week to saidmammalian subject at a does for each administration of about 50 mg-1 gtotal lipid/kg body weight.
 21. The use of claim 20, wherein said doesis about 200-450 mg total lipid/kg body weight.
 22. the use of claim 12,wherein said medicament is suitable for administration by intravenousinjection or intravenous infusion.